Electrostatic and steric interactions determine bacteriorhodopsin single-molecule biomechanics

Abstract

Bacteriorhodopsin (bR) is a haloarchaeal membrane protein that converts the energy of single photons into large structural changes to directionally pump protons across purple membrane. This is achieved by a complex combination of local dynamic interactions controlling bR biomechanics at the submolecular level, producing efficient amplification of the retinal photoisomerization. Using single molecule force spectroscopy at different salt concentrations, we show that tryptophan (Trp) residues use steric specific interactions to create a rigid scaffold in bR extracellular region and are responsible for the main unfolding barriers. This scaffold, which encloses the retinal, controls bR local mechanical properties and anchors the protein into the membrane. Furthermore, the stable Trp-based network allows ion binding to two specific sites on the extracellular loops (BC and FG), which are involved in proton release and lateral transport. In contrast, the cytoplasmic side of bR is mainly governed by relatively weak nonspecific electrostatic interactions that provide the flexibility necessary for large cytoplasmic structural rearrangements during the photocycle. The presence of an extracellular Trp-based network tightly enclosing the retinal seems common to most haloarchaeal rhodopsins, and could be relevant to their exceptional efficiency.

FIGURE 1

AM-AFM height (a) and phase image (b) of PM cytoplasmic (lower) and extracellular (upper) surfaces in liquid. Both images exhibit the well-known hexagonal bR lattice with the cytoplasmic surface allowing resolution of individual proteins while only trimers are visible on the extracellular surface. The side assessment is consistent with previously published high-resolution images of PM surface (84) and with the observed phase shift between the membrane two sides and the mica (23). The image was acquired immediately after several successful unfolding attempts on the membrane extracellular terminus, showing that the AFM tip is still clean. The size is 200 × 200 nm for both images. An example of three-dimensional statistical histogram obtained using the analysis software described in Materials and Methods is shown in panel c. F_unf is the unfolding force associated with a step corresponding to L_out aa extended outside of the membrane (see Fig. 2). The buffer is 30 mM KCl, 10 mM Tris-HCl at pH 8 for panels a and b, and 20 mM KCl, 10 mM Tris-HCl at pH 8 for panel c.

FIGURE 2

Unfolding principle. The AFM tip binds to bR cytoplasmic terminus (a). The tip starts to unfold the protein by stretching part of helix G up to a first unfolding barrier (Lys²¹⁶) (b). When the pulling force is sufficient, the barrier is overcome and the helix can be stretched up to the next unfolding barrier (Trp¹⁸⁹) (c). The effective protein length stretched is L_out+ L_in but the WLC only accounts for L_out. It is therefore necessary to know L_in (calculated from (14) and (16)) to accurately determine the unfolding barrier location (see Appendix A for a detailed example).

FIGURE 3

Histograms representing the number of aa extended out of the membrane with each unfolding step for the different salt concentrations studied (L_out in Fig. 2). The histograms were obtained from individual fitting of each curve unfolding steps with the WLC model (Fig. 4). The steps are represented by Gaussian curves which, at full width at half-maximum, correspond to the fitting standard deviation. Unfolding was carried out from bR C-terminus (a–c) and N-terminus (d–f) independently at each salt concentration investigated. The steps reported in Table 2 were obtained by fitting the histograms with Gaussian distributions. The Gaussian color represents the salt concentration at which the corresponding step has been first reproducibly observed (blue at 20 mM KCl, 10 mM Tris-HCl, pH 8; red at 30 mM KCl, 10 mM Tris-HCl, pH 8; and black at 40 mM KCl, 10 mM Tris-HCl, pH 8). For each histogram, the corresponding top view of a three-dimensional force plot is also presented (the color scale represents the frequency of the reported events).

FIGURE 4

WLC fit (light shaded) of representative unfolding curves from bR C- and N-terminus (solid) for each salt concentration studied. The fits were obtained using the analysis procedure described in Appendix B. The notation used to designate the condition in which each unfolding curve presented was acquired is identical as in Fig. 3. All the KCl solutions are buffered with 10 mM Tris-HCl at pH 8.

FIGURE 5

Scheme of bR aa sequence in the membrane (according to (14)). The Trp residues are boxed; Lys²¹⁶ (linking the retinal via a Schiff base) and the glutamates involved in extracellular ion binding are circled. The helices and the termini are labeled.

FIGURE 6

Scheme of the unfolding barrier positions reported in Table 2. The barriers are symbolized by a sphere or an ellipsoid fitting the residue(s) believed to be responsible for the step observed. At each salt concentration, the steps are classified following the order of the unfolding events (Fig. 3). The color of the arrows indicating the unfolding direction coincides with that of the structural element being unfolded. The color code used for bR helices is shown at the bottom (same color code as for Fig. 2). In each cartoon, the cytoplasmic side of PM is up, and the extracellular side is down. The extraction of both bR termini at 40 mM KCl (Table 2) is not shown.